CHO cells transiently transfected with GHSR1a alone and cotransfected with GHSR1a and MRAP2 were harvested, lysed in HE buffer {10 mM Hepes (pH 7.4), 1 mM EGTA, and protease inhibitor mixture [phenylmethylsulfonyl fluoride (23 μg/ml), Nα-p-tosyl-l-lysine-chloromethyl ketone (21 μg/ml), l-1-p-tosylamino-2-phenylethyl-chloro ketone (21 μg/ml), leupeptin (3.3 μg/ml), and lima bean trypsin inhibitor (3.3 μg/ml)]} using a Parr disruption vessel (Parr Instrument Co.). Cell debris were cleared by centrifugation at 1500g. Membranes were recovered by centrifugation at 100,000g and washed by Dounce homogenization in HE buffer. Membranes were recentrifuged and Dounce-homogenized into membrane storage buffer [HE buffer with 12% (v/v) sucrose] and stored at −80°C. Prepared GHSR1/MRAP2 membranes (10 μg per assay point) were incubated for 5 min with 200 nM Gαq and 500 nM Gβ1γ2 in preincubation buffer [50 mM Hepes (pH 7.4), 1 mM dithiothreitol (DTT), 1 mM EDTA, and BSA (3 μg/ml)]. For agonist assays, membranes were incubated with or without 10 μM ghrelin for 5 min and then with G protein. Endpoint assays were initiated by addition of an equal volume of [35S]GTPγS binding buffer [50 mM Hepes (pH 7.4), 1 mM DTT, 1 mM EDTA, 20 μM GDP, BSA (3 μg/ml), 10 mM MgCl2, 50 mM NaCl, and 2 μM [35S]GTPγS (20,000 to 50,000 cpm/pmol)]. Reactions were performed in triplicate, quenched after 30 min with 20 mM tris (pH 7.7), 100 mM NaCl, 10 mM MgCl2, 1 mM GTP, and 0.08 (m/v) deionized polyoxyethylene 10 lauryl ether C12E10, and then filtered onto Protran BA85 nitrocellulose filters (GE Healthcare). Filters were washed with 20 mM tris (pH 7.7), 100 mM NaCl, and 2 mM MgCl2, dried, and subjected to liquid scintillation counting.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.